Inhibition of KdpFABC by KdpD. KdpFABC constructs were purified from E. coli LB2003 cells, which natively express kdpDE , or from E. coli TK2281 cells, in which the kdpFABCDE operon is deleted. In the latter, KdpD variants were reintroduced as indicated by expression from an orthogonal plasmid. Phosphorylation state of KdpB visualized by phosphoprotein gel stain. The Coomassie stain indicates comparable amounts of protein loaded per lane in the phosphoprotein stain. Full phosphoprotein and Coomassie stains are shown in Supplementary Fig. 1 , successful co-expression of KdpD variants is shown in Supplementary Fig. 2 .
